The present invention relates to the use of 2-amino-thiazoline derivatives of formula (I): 
or pharmaceutically acceptable salts thereof as inhibitors of inducible NO-synthase.
The subject of the invention is the use of 2-amino-thiazoline derivatives of formula (I) and pharmaceutically acceptable salts thereof for the preparation of pharmaceutical compositions intended for preventing and treating diseases in which an abnormal production of nitric oxide (NO) by induction of inducible NO-synthase (NOS-2 ou iNOS) is involved, the pharmaceutical compositions containing the novel 2-amino-thiazoline derivatives and pharmaceutically acceptable salts thereof and the novel derivatives of 2-amino-thiazoline and pharmaceutically acceptable salts thereof.
Nitric oxide (NO) is a diffusable radical involved in many physiological and pathological processes. It is synthesized by oxidation of L-Arginine, a reaction catalyzed by a family of enzymes known as nitric oxide synthases or NO-Synthase (NOS), referenced in the international enzyme nomenclature under the number E.C. 1.14.13.39.
Three NOS isoforms, two of which are constitutive and one inducible, are known:
a neuronal NOS (NOS-1 or nNOS) was originally isolated and cloned from nerve tissue in which it is a constitutive enzyme. The NOS-1 produces NO in response to various physiological stimuli such as the activation of membrane receptors according to a mechanism dependent on calcium and on calmodulin.
an inducible NOS (NOS-2 or iNOS) can be induced in response to immunological stimuli such as, for example, cytokines or bacterial antigens in various cells such as, for example, macrophages, endothelial cells, hepatocytes, glial cells, as well as many other types of cells. This isoform activity is not regulated by calcium. Consequently, once induced, it produces a large amount of NO over prolonged periods.
an endothelial NOS (NOS-3 or eNOS) is constitutive and calcium/calmodulin dependent. It was originally identified in vascular endothelium cells, in which it generates NO in response to physiological stimuli such as the activation of membrane receptors.
The NO produced by the neuronal and endothelial constitutive isoforms (NOS-1 and NOS-3) is generally involved in intercellular signalling functions. For example, the endothelial cells which line the inner wall of blood vessels induce the relaxation of the underlying smooth muscular cells via the production de NO. It thus contributes towards regulating the arterial pressure.
The NO produced in large amount by the inducible isoform NOS-2 is, inter alia, involved in the pathological phenomena associated with acute and chronic inflammatory processes in a large variety of tissues and organs.
An excessive production of NO by induction of NOS-2 thus plays a part in degenerative pathologies of the nervous system such as, for example, multiple sclerosis, focal or global cerebral ischemia, cerebral or spinal trauma, Parkinson""s disease, Huntington""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis, migraine, depression, schizophrenia, anxiety, epilepsy. Similarly, aside the central nervous system, the induction of NOS-2 is involved in many pathologies with inflammatory components such as, for example, diabetes, atherosclerosis, myocarditis, arthritis, arthrosis, asthma, inflammatory bowel disease, Crohn""s disease, peritonitis, gastroesophageal reflux, uveitis, Guillain-Barrxc3xa9 syndrome, glomerulo-nephritis, lupus erythematosus and psoriasis. The NOS-2 was also involved in the growth of certain forms of tumors such as, for example, epitheliomas, adenocarcinomas or sarcomas, and in infections with Gram-positive or Gram-negative intracellular or extracellular bacteria.
In all the situations in which an overproduction of NO is deleterious, it thus appears to be desirable to reduce the production of NO by administering substances capable of inhibiting the NOS-2. However, given the important physiological roles played by the constitutive isoform NOS-3, in particular in regulating the arterial pressure, it is essential that the inhibition of the isoform NOS-2 has the least possible effect on the isoform NOS-3. Actually, it is known that the administration of unselective inhibitors of NOS isoforms leads to vasoconstriction and an increase in arterial pressure (Moncada, S., Palmer, R. M. J. and Higgs, E. A., Biosynthesis of nitric oxide from L-arginine: a pathway for the regulation of cell function and communication, Biochem. Pharmacol., 1989, 38: 1709-1715). These effects on the cardiovascular system are deleterious since they reduce the supply of nutrients to the tissues. Consequently, the present invention relates to compounds whose inhibitory activity with respect to NOS-2 is significantly higher than their inhibitory activity with respect to NOS-3.
Thiazoline-based NOS inhibitors are described in particular in patent applications WO94/12165, WO95/11231 and WO96/14842.
The present invention relates to the use of 2-amino-thiazoline derivatives of formula (I) in which:
either Y is a methylene (CH2) and X is chosen from the following groups: O, NH, N-(C1-C4)alkyl, N-Bn, N-Ph, N-(2-Py), N-(3-Py), N-(4-Py), N-2-pyrimidyl, N-5-pyrimidyl, S, SO, SO2, CH2 or CHPh;
or Y is a carbonyl (Cxe2x95x90O) and X is chosen from the following groups: NH, N-Ph, N-(2-Py), N-(3-Py), N-(4-Py), N-2-pyrimidyl, N-5-pyrimidyl for the preparation of medicinal products for preventing and treating diseases in which an abnormal production of nitric oxide (NO) by induction of inducible NO-synthase (NOS-2 or iNOS) is involved.
In the above definitions and in those which follow, the alkyl radicals contain 1 to 4 carbon atoms in a straight or branched chain. The abbreviations Bn, Py, Ph mean respectively benzyl, pyridyl, phenyl.
The compounds of formula (I) contain one or more asymmetric carbons and can thus be in racemic form or in the form of enantiomers and diastereoisomers; these also form a part of the invention as well as the mixtures thereof.
Moreover, the compounds of formula (I) can be in the tautomeric form (Ia): 
These tautomers also form a part of the invention.
Among the compounds of formula (I) useful according to the invention, mention may be made of the following compounds:
4-(morpholin-4-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine,
4-(piperazin-1-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine, and
4-(4-methyl-piperazin-1-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine,
the racemic mixtures, enantiomers, diastereoisomers, tautomers thereof, as well as the pharmaceutically acceptable salts thereof.
Among the compounds useful according to the invention and particularly prefered, mention may be made of the following compound:
4-(4-methyl-piperazin-1-ylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine,
the racemic mixtures, enantiomers, tautomers thereof, as well as the pharmaceutically acceptable salts thereof.
The invention also relates to the pharmaceutical compositions containing, as active principle, a derivative of formula (I) for which either Y is a methylene (CH2) and X is chosen from the following groups: O, NH, N-(C1-C4)alkyl, N-Bn, N-Ph, N-(2-Py), N-(3-Py), N-(4-Py), N-2-pyrimidyl, N-5-pyrimidyl, S, SO, SO2, CH2 or CHPh; or Y is a carbonyl (Cxe2x95x90O) and X is chosen from the following groups: NH, N-Ph, N-(2-Py), N-(3-Py), N-(4-Py), N-2-pyrimidyl, N-5-pyrimidyl as well as the racemic mixtures, enantiomers, diastereoisomers, tautomer thereof, and pharmaceutically acceptable salts thereof.
The compounds of formula (I) can be prepared by cyclization of a derivative of formula (II): 
in which X and Y have the same meaning as in formula (I).
This cyclization is generally carried out using an acid such as hydrochloric acid, in aqueous medium, at a temperature of about 100xc2x0 C. 6N hydrochloric acid is generally used.
The derivatives of formula (II) can be obtained according to the following reaction schemes: 
in these formulae X and Y have the same meanings as in formula (I), Ra is a protecting group of the amine function such as those described by T. W. GREENE, Protective groups in Organic Synthesis, J. Wiley-Interscience Publication (1991), preferably an acetyl or tert-butyloxycarbonyl radical, and Rb is a (C1-C4) alkyl or alkoxycarbonyl radical, preferably methyl, ethyl or isobutyloxycarbonyl.
The reaction a is generally carried out in the presence of a Lewis acid such as the iron trichloride (III), in an inert solvent such as dichloromethane or acetonitrile, at a temperature of between 10xc2x0 C. and the boiling point of the reaction medium. When X represents NH, X can be protected by a protecting group of the amine function such as described by T. W. GREENE, Protective Groups in Organic Synthesis, J. Wiley-Interscience Publication (1991), preferably using a tert-butoxycarbonyl radical.
The reduction reaction b is preferably carried out using a hydride such as sodium borohydride or lithium aluminum hydride in a (C1-C4) aliphatic alcohol or tetrahydrofuran, at a temperature of between 0xc2x0 C. and 30xc2x0 C.
The deprotection reaction c for the compounds in which Ra is a protecting group of the amine function is carried out by any deprotection method known to those skilled in the art and in particular those described by T. W. GREENE, Protective Groups in Organic Synthesis, J. Wiley-Interscience Publication (1991). Preferably when the protecting group is an acetyl radical, this reaction is carried out using aqueous hydrochloric acid at a temperature of about 100xc2x0 C. When the protecting group is a tert-butoxycarbonyl radical, this reaction is carried out using hydrochloric acid in dioxane, at a temperature of about 20xc2x0 C.
The reaction d is carried out by the action of tert-butyl isothiocyanate, in an inert solvent such as (C1-C4) aliphatic alcohol (preferably methanol or ethanol), optionally in the presence of a tertiairy amine such as triethylamine, at a temperature between 20xc2x0 C. and the boiling point of the reaction medium.
The compounds of formula (I) in which X represents either SO, or SO2 can be obtained by direct oxidation of the compound of formula (I) in which X represents S. This oxidation is carried out according to the known methods of oxidation of organosufur compounds, such as described by M. HUDLICKY, Oxidation in Organic Chemistry, ACS Monograph, 186, 252-263 (1990). For example, it is carried out by the action of an organic peracid or organic peracid salt (percarboxylic or persulfonic acid, in particular perbenzoic acid, 3-chloro-perbenzoic acid, 4-nitroperbenzoic acid, peracetic acid, pertrifluoroacetic acid, performic acid, monoperphthalic acid) or a mineral peracid or mineral peracide salt (for example, periodic or persulfuric acid), in an inert solvent such as a chlorine solvent (for example, trichlorethane or dichloromethane), at a temperature of between 0xc2x0 C. and 20xc2x0 C. The hydrogen peroxide or periodate (sodium periodate, for example), in an inert solvent such as (C1-C4) aliphatic alcohol, water or a mixture of these solvents, at a temperature between 0xc2x0 and 20xc2x0 C. can also be used. These products can also be prepared from the corresponding compounds of formula (II), obtained according to the following reaction schemes: 
The oxidation reaction a is carried out according to the known methods of oxidation of organosulfur compounds as described above.
The deprotection reaction b for the compounds in which Ra is a protecting group of the amine function is carried out by any method of deprotection known by those skilled in the art and particularly those described by T. W. GREENE, Protective Groups in Organic Synthesis, J. Wiley-Interscience Publication (1991). Preferably when the protecting group is an acetyl radical, this reaction is carried out using aqueous hydrochloric acid, at a temperature of about 100xc2x0 C. When the protecting group is a tert-butyloxycarbonyl radical, this reaction is carried out using an hydrochloric acid in dioxane, at a temperature of about 20xc2x0 C.
The reaction c is carried out by the action of tert-butyl isothiocyanate, in an inert solvent such as (C1-C4) aliphatic alcohol (preferably methanol or ethanol), optionally in the presence of a tertiary amine such as triethylamine, at a temperature of between 20xc2x0 C. and the boiling point of the reaction medium.
The compounds of formula (I) are isolated and can be purified by the usual known methods, for example crystallization, chromatography or extraction.
The enantiomers of the compounds of formula (I) can be obtained by resolving the racemic mixtures, for example by chromatography on a chiral column according to PIRCKLE W. H. et al., Asymmetric Synthesis, Vol. 1, Academic Press (1983) or by formation of salts or by synthesis from chiral precursors. The diastereoisomers can be prepared according to the known conventional methods (crystallization, chromatography or from chiral precursors).
The compounds of formula (I) can optionally be converted to addition salts with a mineral or organic acid by the the action of such an acid in an organic solvent such as an alcohol, a ketone, an ether or a chlorinated solvent. These salts also form a part of the invention.
Examples of pharmaceutically acceptable salts which may be mentioned are the following salts: benzenesulfonate, hydrobromide, hydrochloride, citrate, ethanesulfonate, fumarate, gluconate, iodate, isethionate, maleate, methanesulfonate, methylenebis-xcex2-oxynaphtoate, nitrate, oxalate, pamoate, phosphate, salicylate, succinate, sulfate, tartrate, theophyllinacetate and p-toluenesulfonate.
The compounds of formula (I) are inhibitors of NO-synthase inducible or NO-synthase of type 2 (NOS-2) and are thus useful for preventing and treating disorders associated with an excessive NO production such as mutiple sclerosis, focal or global cerebral ischemia, cerebral or spinal trauma, Parkinson""s disease, Huntington""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis, migraine, depression, schizophrenia, anxiety, epilepsy, diabetes, atherosclerosis, myocarditis, arthritis, arthrosis, asthma, inflammatory bowel disease, Crohn""s disease, peritonitis, gastro-esophageal reflux, uveitis, Guillain-Barrxc3xa9 syndrome, glomerulo-nephritis, lupus erythematosus and psoriasis, the growth of certain forms of tumors such as for example epitheliomas, adenocarcinomas or sarcomas, and in infections with Gram-positive or Gram-negative intracellular or extracellular bacteria.
Their activities as inhibitors of NOS-2 and NOS-3 were determined by measuring the conversion of [3H]-L-arginine into [3H]-L-citrulline with, respectively, a NOS-2 enzymatic fraction extracted from the lungs of rats or mices pretreated with lipopolysaccharides (10 mg/kg i.p. 6 hours before collecting the tissue) and with a commercial preparation of recombinant bovine NOS-3. The compounds were incubated for 20 to 30 minutes at 37xc2x0 C. in the presence of 5 xcexcM (for NOS-2 activity) or 10 xcexcM (for NOS-3 activity) of [3H]-L-arginine, 1 mM of NADPH, 15 xcexcM of tetrabiopterine, 1 xcexcM of FAD, 0.1 mM of DTT in a HEPES buffer (50 mM, pH 6.7) containing 10 xcexcg/ml of calmodulin and 1.25 mM of CaCl2 when the NOS-3 activity was measured. The incubation was stopped by adding cold HEPES buffer (100 mM, pH 5.5) containing 10 mM EGTA and 500 mg of cationic ion-exchange resin (AG50W-X8, counter-ion:Na+) to separate the [3H]-L-arginine from the [3H]-L-citrulline. After separation of the phases by settling for 5 min, the radioactivity remaining in the liquid phase was measured in a scintillation counter in the presence of a suitable scintillation liquid. The yield for the recovery of the formed L-[3H]citrulline was able to be estimated using L-[ureido-14C]-citrulline as external standard.
The NOS-2 or NOS-3 activity was expressed in picomole(s) of [3H]-L-citrulline formed per minute and per milligram of protein contained in the rection medium.
In this test on the enzyme NOS-2, the IC50 value for the compounds of formula (I) is less than or equal to 10 xcexcM.
The selectivity is measured by the IC50 NOS-3/IC50 NOS-2 ratio. This selectivity is greater than 45.
The compounds of formula (I) are of low toxicity. Their LD50 is greater than 40 mg/kg via cutaneous route in mice.
The following examples illustrate the invention.